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4 protocols using lf qc0103

1

Western Blot Analysis of Lung Tissue

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For western blot analysis, proteins collected from whole lung tissue were separated by SDS-PAGE in a 15% polyacrylamide gel for 3 h. The separated proteins were then transferred to PVDF membranes (Bio-Rad, USA) for 1 h at 90 volts. After blocking the membranes overnight at 4 °C with 5% skim milk, they were probed using anti-GAPDH (Santa Cruz Biotechnology, LF-PA0018), polyclonal mouse anti-NF-κB (Santa Cruz Biotechnology, SC-71675), monoclonal mouse anti-TNF-α (ABfrontier, AB1793), polyclonal mouse anti-IL-1β (ABfrontier, AB1413), polyclonal mouse anti-Cathelicidin (ABfrontier, AB93357) and polyclonal mouse anti-PGC-1 (Millipore AB3242). The membranes were then washed with TBST buffer and incubated with secondary antibodies (goat anti-mouse IgG (HRP) LF-SA5001-conjugated). Finally, the blots were developed using a western blot detection kit (LF-QC0103, Abfrontier)74 (link).
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2

Western Blot Analysis of Cell Proteins

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We blocked blotted membranes with blocking buffer (5% bovine serum albumin (BSA) in Tris-buffered saline with 0.05% tween-20 (TBST)) for 1 hat room temperature, then incubated with primary antibodies (goat-anti E-cadherin, AF-748, R&D Systems, Minneapolis, MN, USA, 1:2000, mouse-anti AQP5, sc-514022, Santa Cruz Biotechnology, Delaware Avenue Santa Cruz, CA, USA, 1:2000, mouse-anti β-actin, A2228, Sigma-Aldrich, 1:2000) for 16–21 hat 4 C. After washing with TBST 10 min three times, and we incubated the membranes with HRP-conjugated secondary antibodies (goat-anti mouse, 115-035-141, 1:1000; donkey-antigoat, 705-035-147, 1:1000, Jackson ImmunoResearch, West Grove, PA, USA) for 1.5 hat room temperature. The immunoblotting was visualized using the enhanced chemiluminescence reagent (ECL; AbFrontier, LF-QC0103, Seoul, South Korea). When we used the membranes more than once, we removed antibodies with a low-pH stripping buffer (pH 2.2; 200 mM glycine, 1% SDS, 0.03% Tween-20) for 5–10 minat RT and then re-start the blocking step.
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3

Temporal Bone Protein Analysis

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At days 0, 1, 3 and 7, animals were euthanized with a urethane intraperitoneal injection (1 g/kg), and both temporal bones were immediately removed. Two cochleae were homogenized in a tube containing ice-cold lysis buffer (100 mM Tris, pH 7.4, 200 mM NaCl, 1% NP-40, 10 mM MgCl2) with protease inhibitors. Western blotting was performed, as described previously.17 (link) In brief, lysates were centrifuged for 20 min at 13 000 r.p.m. at 4 °C; in addition, proteins in the supernatant were separated using 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Blots were incubated overnight with the primary antibodies against 4-HNE (1 : 1000 dilution), BDNF (ab6201, 1 : 1000 dilution; Abcam) and NT-3 (ab65804, 1 : 1000 dilution; Abcam). After rinsing in TBST, the membranes were incubated with the HRP-conjugated secondary antibody (81–1620, A16104, G21040; 1 : 5000; Invitrogen) for 1 h. Bands were detected by chemiluminescence (LF-QC0103, Ab Frontier, Seoul, Korea). Further, blots were stripped and reprobed with actin (SC-1616, Santa Cruz Biotechnology), thereby serving as a protein loading control. Band intensities were quantified using the ImageJ software (NIH).
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4

Western Blot Analysis of Cell Signaling Proteins

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Cells were lysed in a lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate and 0.1% SDS) with a protease inhibitor cocktail (#P2714, Sigma-Aldrich). Proteins were resolved on SDS–polyacrylamide gels and transferred to PVDF membranes, which were then probed with specific antibodies. The protein-antibody complexes were detected by enhanced chemiluminescence (#LF-QC0103, AbFrontier, South Korea). Specific antibodies for COX-2 (1:500; #sc-1745), p21 (1:500; #sc-397), p16 (1:50; #sc-28260) and β-actin (1:20,000; #sc-81178) were purchased from Santa Cruz Biotechnology. Antibodies for p53 (1:500; #2524) and Ras (1:1000; #OP40) were purchased from Cell Signaling Biotechnology and Calbiochem, respectively. Blots were cut prior to hybridization with the specific antibodies and therefore the whole length blots were not provided.
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